DESCRIPTION: Adenylate cyclase (AC) toxin is a novel protein bacterial toxin produced by Bordetalla pertussis, the causative agent of whooping cough. The sequence of this toxin is known and palimiolyation of a single Lys residue has been demonstrated to be essential for toxin activities. Nevertheless, little is known about mechanisms for toxin-mediated processes or their functional interrelationships. This proposal will focus on the individual steps involved in AC toxin action. Assays will be adapted or developed to enable the isolation and characterization of each individual step in the pathway. The methodologic approaches will include use of intrinsic tryptophan fluorescence fluoresecence energy transfer, lipid bilayer and whole cell patch-clamp conductance, electron microscopy and x-ray diffraction. The functional studies will be complemented with th use of random and site-specific mutagenesis to generate mutants of AC toxin that are defective in toxin and/or hemolysin activities. Similarly, an in vitro acylation will provide a mechanism for preparation of toxins with other than palmitoyl groups on LYs983, in order to determine the role of acylation. Additional structural data will be collected with CD spectroscopy an x-ray diffraction studies to develop a better understanding of the actions of AC toxin.